Transcript Plant Breeding and Plant Biotechnology

Plant Tissue Culture
Media
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Logical Basis
For healthy and vigorous growth, intact plants need to take up
from soil of an essential elements:
 Relatively large amount of some inorganic elements (major
plant nutrition)
a. Nitrogen (N)
b. Calcium (Ca)
c. Magnesium (Mg)
d. Potassium (K)
e. Phosphorus (P)
f. Sulphur (S)
 Small quantities of other elements (minor plant
nutrient/trace elements)
a. Iron (Fe)
b. Chlorine (Cl)
c. Zinc (Zn)
d. Copper (Cu)
e. Nickel (Ni)
f. Sodium (Na)
g. Manganese (Mn)
h. Boron (B)
i. Molybdenum (Mo)
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Essential elements
(Epstein, 1971)
1. A plant grown in a medium adequately
purged of that elements, failed to
grow properly or to complete its life
cycle
2. It is a constituent of a molecule that is
known to be an essential metabolite
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Functions of medium
Provide water
Provide mineral nutritional needs
Provide vitamins
Provide growth regulators
Provide amino acids
Provide sugars
Access to atmosphere for gas exchange
Removal of plant metabolite waste
Plant tissue culture media
1. Macronutrients (always employed)
2. Micronutrients (nearly always employed, although sometimes just
one element, iron, has been used)
3. Vitamins (generally incorporated , although the actual number of
compounds added, varies greatly)
4. Amino acids and other nitrogen supplements (usually omitted, but
sometimes used with advantage)
5. Sugar (nearly always added, but omitted for some special purposes)
6. Undefined supplements (which, when used, contribute some above
components, and also plant growth substances or regulants)
7. Buffers (have seldom been used in the past, but recently suggest
that the additions of organic acids or buffers could be beneficial in
some circumstances)
8. A solidifying agent (used when a semi solid medium is required)
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Macronutrient
1. Macronutrients for plant tissue culture are provided from salt,
however plant absorb entirely as ions
2. Nitrogen is mainly absorbed in the form of ammonium or nitrate
3. Phosphorus as the phosphate ions
4. Sulphur as sulphate ions
5. The most important step in deriving medium is the selection of
macronutrient ions in the correct concentration and balanced
6. The salts normally used to provide macroelements also provide
sodium and chlorine, however, plant cell tolerate high
concentration of both ions without injury, these ions are
frequently given little importance when contemplating media
changes
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Quantity of the Macronutrient
Nitrogen
1. It is essential to plant life
2. Both growth and morphogenesis is markedly influenced by the
availability of nitrogen and the form in which it is presented
3. Most media contain more nitrate than ammonium ions. Most
intact plants, tissues and organ taken up nitrogen effectively, and
grow more rapidly on nutrient solutions containing both nitrate
and ammonium ions
4. Nitrate has to be reduced to ammonium before being utilized
biosynthetically
5. Ammonium in high concentration is latent toxic
6. For most type of culture, nitrate needs to be presented together
with the reduced form of nitrogen and tissue will usually fail to
grow on a medium with nitrate as the only nitrogen source
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NH4+ and NO3- Regulate Medium pH
and Root Morphogenesis of Rose Shoots
Amino acids
1. Amino acids can be added to satisfy the requirement for
reduced nitrogen, but as they are expensive to purchase, they
will only be used on media for mass propagation where this
results in improved result
2. A casein hydrolysate, yeast extract which mainly consist of a
mixture of amino acids substantially increased the yield of
callus
3. Organic supplements have been especially beneficial for
growth or morphogenesis when cells were cultured on media
which do not contain ammonium ions
4. Glycine os an ingredient of many media. It is difficult to find
hard evidence that glycine is really essential for so many tissue
culture, but possible it helps to protect cell membranes from
osmotic and temperature stress
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Amino Acids
The most common sources of organic nitrogen used in
culture media are amino acid mixtures.
 Its uptake more rapidly than in organic amino acids
. (e.g., casein hydrolysate), L-glutamine, L-asparagine,
and adenine.
When amino acids are added alone, they can be inhibitory
to cell growth.
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Beneficial effects of amino
acids
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Rapid growth
Protoplast cell division
Conservation of ATP
AS chelating agent
Enhanced nitrogen assimilation
Not toxic as ammonium
As buffer
Micronutrients
1. Plant requirement for microelement have only been elucidated in
the 19th century
2. In the early of 20th century, uncertainty still existed over the
nature of the essential microelements
3. many tissue undoubtedly grown successfully because they were
cultured on media prepared from impure chemicals or solidified
with agar which acted as a micronutrient source
4. In the first instance, the advantage of adding micronutrients was
mainly evaluated by their capability to improve the callus growth
or root culture
5. Knudson (1922) incorporated Fe and Mn on very successful
orchid seed media
6. Heller (1953) was first well demonstrated the advantages of
microelement on tissue culture media
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Quantity of the Micronutrient
MS medium was formulated from the ash content of tobacco callus.
The higher concentration of salts substantially enhanced cell division
Chelating agent
1. Some organic compounds are capable of forming complexes with
metal cations, in which the metal is held with fairly tight chemical
bonds
2. Metal can be bound (sequestered) by a chelating agent and held in
solution under conditions where free ions would react with anions
to form insoluble compounds, and some complexes can be more
chemically reactive than the metals themselves
3. Chelating agents vary in their sequestering capacity according to
chemical structure and their degree of ionisation, which changes
with pH of the solution
4. Naturally –occurring compounds can act as chelating agents such
as proteins, peptides, carboxylic acids and amino acids
5. There are also synthetic chelating agents with high avidity for
divalent and trivalent ions
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Chelating agents
No. Chelating agents Chemical names
1.
EDTA
Ethylenediamine tetra acetic acid
2.
EGTA
Ethyleneglycol-bis(2aminoethylether) tetra acetic acid
3.
EDDHA
Ethylenediamine-di(ohydroxyphenyl) acetic acid
4.
DTPA
Diethylenetriaminepentaacetic acid
5.
DHPTA
1,3 diamino-2-hydroxypropanetetra acetic acid
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Iron and iron chelates
1. A key properties of iron is its capacity to be oxidized easily from
the ferrous (Fe(II)) to the ferric (Fe(III)) state and for ferric
compounds to be readily reduced back to the ferrous form
2. Iron is primarily used in the chloroplasts, mitochondria and
peroxisomes for effecting oxidation/reduction reaction
3. It is a component of ferredoxin proteins which function as
electron carriers in photosynthesis
4. Iron is an essential micronutrient for plant tissue culture and can
be taken up as either ferrous or ferric ions
5. Iron may not be available to plant cells, unless the pH falls
sufficiently to bring free ions back to solutions
6. Iron can be chelated with EDTA
7. The addition of Fe-EDTA chelate greatly improved the
availability of the element
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Carbon Source
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Most plant tissue cultures are not highly autotrophic due to limitation
of CO2. Therefore, sugar is added to the medium as an energy source.
Sucrose is the most common sugar added, although glucose, fructose,
manitol and sorbitol are also used in certain instances.
The concentration of sugars in nutrient media generally ranges from 20
to 40 g/l.
Sugars also contribute to the osmotic potential in the culture
The presence of sucrose specifically inhibits chlorophyll formation and
photosynthesis, making autotrophic growth less feasible
Sucrose in the culture media is usually hydrolyzed totally or partially
into the component monosaccharides glucose and fructose
The general superiority of sucrose over glucose may be on account of
the more effective translocation of sucrose to apical meristems
Organic supplement
a)
Vitamins:
Only thiamine (vitamin B1) is essential for most
plant cultures, it is required for carbohydrate
metabolism and the biosynthesis of some amino
acids
 Thiamine
(vitamin B1)
Essential as a coenzyme in the citric acid cycle
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Nicotinic acid (niacin) and pyridoxine (B6)
Organic supplement
b)
Myo-inositol
Although it is not essential for growth of many plant species, its
effect on growth is significant.
Part of the B complex, in phosphate form is part of cell
membranes, organelles and is not essential to growth but
beneficial
c)
Complex organics
Such as coconut milk, coconut water, yeast extract, fruit juices
and fruit pulps.
Physical support agents
A. Gelling agents
When semi-solid or solid culture media are required,
gelling agents are used.
An example:
Agar, agarose, gelrite, phytagel
B. Structural supports
Filter paper bridges, liquid permeable
support systems
membrane
Agar
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Agar is the most commonly used gelling agent
It is a natural product extracted from species of red algae,
especially Gelidium amansii
It is synthetic polysaccharide gelling agents
Agar consists of 2 components
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Agarose is an alternating D-galactose and 3,6-anhydro-L-galactose
with side chains of 6-methyl-D-galactose residues (50 -90%).
Agaropectin is like agarose but additionally contains sulfate ester side
chains and D-glucuronic acid.
Agar tertiary structure is a double helix the central cavity of
which can accommodate water molecules
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Advantages:
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Agar is an inert component, form a gel in water that melt at
100 ° C and solidify at nearly 45 ° C
Concentrations commonly used in plant culture media range
between 0.5% and 1%
If necessary, agar can be washed to remove inhibitory organic
and inorganic impurities.
Gels are not digested by plant enzymes
Agar does not strongly react with media constituent
Disadvantages:
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Agar does not gel well under acidic conditions (pH <4.5).
The inclusion of activated charcoal in media may also inhibit
gelling of agar.
Agarose
It is extracted from
agar leaving behind
agaropectin and its
sulfate groups.
 It is used when the
impurities of agar are
a major disadvantage.
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Gelrite™
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Gelrite consists of a polysaccharide
produced
by
the
bacterium
Pseudomonas elodea.
It gives clear-solidified medium that
leads to detection of contamination at
an early stage.
Gelrite requires more stirring than
agar.
Concentration of divalent cations
such as calcium and magnesium must
be within the range of 4-8 mM/L or
the medium will not solidify
Phytagel™
It is an agar substitute produced from a bacterial
substrate composed of glucuronic acid,
rhamnose and glucose.
 It produces a clear, colorless, high-strength gel,
which aids in detection of microbial
contamination.
 It is used at a concentration of 1.5-2.5 g/L.
 It should be prepared with rapid stirring to
prevent clumping.
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Commercial Media Formulations
• Murashige and Skoog (MS)
• Linsmaier and Skoog (LS)
• White Medium
• Gamborg medium
• Schenk and Hildebrandt medium
• Nitsch and Nitsch Medium
• Lloyd and McCown Woody plant medium
• Knudson’s medium
Hormone
(the Greek word hormaein, meaning "to excite").
Small organic molecule that elicits a physiological response
at very low concentrations
Chemical signals that coordinate different parts of the
organism
Internal and external signals that regulate growth are
mediated, at least in part, by growth-regulating
substances, or hormones
Plant Hormone
Plant hormones differ from animal hormones in that:
 No evidence that the fundamental actions of plant and
animal hormones are the same.
 Unlike animal hormones, plant hormones are not
made in tissues specialized for hormone production.
(e.g., sex hormones made in the gonads, human
growth hormone - pituitary gland)
 Unlike animal hormones, plant hormones do not have
definite target areas (e.g., auxins can stimulate
adventitious root development in a cut shoot, or shoot
elongation or apical dominance, or differentiation of
vascular tissue, etc.).
Characteristics
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Synthesized by plants.
Show specific activity at very low concentrations
Display multiple functions in plants.
Play a role in regulating physiological phenomena in
vivo in a dose-dependent manner
They may interact, either synergistically or
antagonistically, to produce a particular effect.
Auxin
Cytokinin
Gibberelin
Classification of
PGRs
Abscisic acid
Ethylene
Other
Jasmonates
Salicylic acid
Brassinosteroids
Auxins
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Absolutely essential (no mutants known)
One compound: Indole-3-acetic acid.
Many synthetic analogues:
NAA, IBA, 2,4-D, 2,4,5-T, Picloram
Cheaper & more stable
Generally growth stimulatory.
Promote rooting
Stimulate cell elongation
Increase the rate of transcription
Mediate the response of bending in response to gravity or light
Produced in meristems, especially shoot meristem and transported
through the plant in special cells in vascular bundles.
Cytokinins
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Absolutely essential (no mutants known)
Natural compound: Zeatin, 2-isopentyl adenine (2iP)
Synthetic analogues: Benyzladenine (BA), Kinetin.
Stimulate cell division (with auxins).
Promotes formation of adventitious shoots
Stimulate cell division
Stimulate dark germination
Stimulate leaf expansion
Produced in the root meristem and transported throughout the
plant as the Zeatin-riboside in the phloem.
Auxin and Cytokinin Ratio
Gibberellins (GA’s)
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A family of over 70 related compounds, all forms of Gibberellic
acid and named as GA1, GA2.... GA110.
Commercially, GA3 and GA4+9 available.
Stimulate etiolation of stems.
Help break bud and seed dormancy.
Stimulate stem elongation by stimulation cell division and
elongation
Stimulate germination of pollen
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Produced in young leaves
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Abscisic Acid (ABA)
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Only one natural compound.
Promotes leaf abscission and seed dormancy.
Plays a dominant role in closing stomata in response to water
stress
Involved in the abscission of buds, flower and fruits
Inhibit cell division and elongation
Has an important role in embryogenesis in preparing embryos
for desiccation.
Helps ensure ‘normal’ embryos.
Ethylene
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Gas - diffuses through tissues
Stimulates abscission and fruit ripening
Used in commercial ripening for bananas & green picked fruit
Involved in leaf abscission & flower senescence
Primarily synthesized in response to stress
Regulate cell death programming
Brassinosteroids
Promote shoot elongating
 Inhibit root growth
 Promote ethylene biosynthesis
 Enhance resistance to chilling, disease and
herbicides
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Salicylic acid
Promote flowering
Stimulate plant pathogenesis protein
production
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Jasmonate
Play an important role in plant defence
mechanisms
Jasmonate
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Play an important role in plant defence mechanisms
Explants
Sterile pieces of a whole plant from which cultures
are generally initiated
Types of explant:
Generally all plant cells can be used as an explant,
however young and rapidly growing tissue (or
tissue at an early stage of development) are
preferred.
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Root tip:
Root cultures can be established from explants of the root tip of
either primary or lateral roots.
Shoot tip:
The shoot apical meristem from either axillary or adventitious buds
can be cultured in vitro.
Embryo:
Both immature and mature embryos can be used as explants to
generate callus cultures or somatic embryos.
Immature, embryo-derived callus is the most popular method of
monocot plant regeneration.
Haploid tissue
Male gametophyte (Pollen in anthers) or female gametophyte (the
ovule) can be used as an explant.
Haploid tissue cultures can produce haploid or di-haploid plants due
to doubling of chromosomes during the culture periods.
Callus
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Definition: It is an unspecialized and
unorganized, growing and dividing mass
of cells, produced when explants are
cultured
on
the
appropriate
solid
medium, with both an auxin and a
cytokinin and correct conditions.
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During callus formation there is some
degree of dedifferentiation both in
morphology and metabolism, resulting
in the lose the ability to photosynthesis.
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This necessitates the addition of other components e.g.: vitamins and, a
carbon source to the culture medium, in addition to the usual mineral
nutrients.
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Habituation: it is the lose of the requirement for auxin and/or cytokinin
by the culture during long-term culture.
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Callus cultures may be compact or friable.
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Compact callus shows densely aggregated cells
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Friable callus shows loosely associated cells and the callus becomes
soft and breaks apart easily.
Cell-suspension cultures
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When friable callus is placed into the appropriate liquid medium and
agitated, single cells and/or small clumps of cells are released into
the medium and continue to grow and divide, producing a cellsuspension culture.
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The inoculum used to initiate cell suspension culture should neither
be too small to affect cells numbers nor too large too allow the
build up of toxic products or stressed cells to lethal levels.
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Cell suspension culture techniques are very important for plant
biotransformation and plant genetic engineering.